EP0886679B1 - Parapoxviruses containing foreign dna, their production and their use in vaccines - Google Patents

Abstract

The present invention relates to recombinantly prepared parapoxviruses which carry, in their genomes, deletions or insertions in the form of foreign hereditary information and contain hereditary information, to the preparation of such constructs and to their use in vaccines.

Description

The present invention relates to recombinant parapoxviruses, their preparation, vaccines and immunomodulators containing them.

The genetically modified parapoxviruses according to the invention carry deletions and / or insertions in their genome. The deletion of genomic sections of the Parapockenviren and / or the insertion of foreign DNA can lead to the reduction or loss of their pathogenicity (attenuation). Insertion inserts genetic information from pathogens or biologically active substances into the genome of the parapox viruses. These foreign genetic information is expressed as part of the recombinant parapochenviruses, for example in cell cultures, tissues or in intact organisms.

The recombinant parapoxviruses produced according to the invention are e.g. used in vaccines or immunomodulators. Expression of the foreign DNA in the genome of the parapoxviruses triggers e.g. in the vaccine a defense reaction against the pathogens, which are represented by the foreign genetic information. In addition, the non-specific immunity of the vaccine can be stimulated. (In the following, the term parapox viruses is abbreviated by PPV.)

PPV itself can have an immunomodulatory effect as it stimulates non-pathogen-specific immune reactions in the organism. Parapox virus preparations, for example, are used successfully in veterinary medicine to increase general defenses

Vaccines which are pathogen-specific require several days to weeks depending on the antigen for the establishment of the protection, but then provide a long, lasting from months to years protection.

Vaccines produced on the basis of recombinant parapoxviruses can thus be used as biological products for improved control of infectious diseases, since they both build a long-lasting, pathogen-specific immunity and induce a very rapidly occurring pathogen-specific protection in the organism.

The combination of the immunostimulatory properties of PPV with the expression of foreign antigens which induce homologous and / or heterologous pathogen-specific protection is novel. This allows the production of products that provide both rapid onset, broad pathogen-specific protection against infection and provide long-lasting, pathogen-specific infection protection.

• Parapoxvirus ovis (auch Ecthyma Contagiosum Virus, Virus der Kontagiösen Pustulardermitis oder Orf Virus genannt), das als Prototyp des Genus gilt,
• Parapoxvirus bovis 1 ( auch Bovine Papulöse Stomatitis Virus oder Stomatitis Papulosa Virus genannt) und
• Parapoxvirus bovis 2 (auch Euterpocken Virus, Paravaccinia Virus, Pseudokuhpocken Virus oder Melkerknoten Virus genannt).

The family of vertebrate pox viruses (Chordopoxvirinae) is divided into separate, independent genera. The present invention relates to the genus of PPV, which differ both structurally and genetically from the remaining poxviruses. The PPV are divided into three different species (Ref. # 1):

• Parapoxvirus ovis (also called Ecthyma Contagiosum Virus, Contagious Pustulardermitis Virus or Orf Virus), which is considered a prototype of the genus,
• Parapoxvirus bovis 1 (also called bovine papular stomatitis virus or stomatitis papulosa virus) and
• Parapoxvirus bovis 2 (also called udderpox virus, paravaccinia virus, pseudo-chickenpox virus or milking knot virus).

Also described are parapoxvirus representatives isolated from camels, red deer, chamois, seals, seals and sea lions. Whether it is a stand-alone species within the genus Parapox viruses or isolates of the species described above, is not yet finally resolved.

PPV infections can cause local diseases in animals and humans (zoonotic agents). Ref. # 1 gives an overview of the clinical pictures described so far. The fight against the diseases is possible through the use of prophylactic measures, such as vaccines. However, the vaccines available so far, which were developed exclusively on the basis of Parapopxvirus ovis, show unsatisfactory activity (Ref. # 2).

The object of the invention is to utilize the PPV as a carrier (vector) of foreign genetic information which is expressed.

Vectors based on avipox, raccoonpox, capripox, swinepox or vaccinia virus have already been described as vectors for the expression of foreign genetic information. The insights gained can not be transferred to PPV. As comparative studies have shown, there are morphological, structural and genetic differences between the individual genera of the poxviruses. For example, the PPV can be differentiated from the other genera of the pox viruses with serological methods, which is due to different protein patterns and thus to different genetic information. For example, some representatives of the poxviruses have the ability to agglutinate erythrocytes. This activity is mediated by a surface protein, the so-called haemagglutinin (HA). PPVs do not have this activity.

The knowledge of the organization of the genome of PPV is currently limited to the size of the genome, the GC content of the nucleic acid, comparative restriction enzyme analyzes, the cloning of individual genome fragments, sequence analysis of subdomains and the related, preliminary description of individual genes (for review Ref # 1, Ref # 5, Ref # 6).

At Vaccinia known insertion sites can not currently be used because of their lack or undetermined existence in PPV.

For example, attempts to identify the gene for thymidine kinase in the genome of the PPV and to use it as an insertion site, as in orthopoxviruses, gave no results. Although Mazur and coworkers (Ref. # 3) describe the identification of a genome portion of PPV that is said to be similar to the thymidine kinase gene of vaccinia virus (an orthopox virus), extensive in-depth studies have failed to confirm the existence of such a gene in PPV. Other authors (Ref. # 1) could not find a thymidine kinase gene in PPV. In vaccinia virus, the gene for HA is used as an insertion site for foreign DNA. PPVs do not have this activity as described above.

Robinson and Lyttle mentioned alternative insertion sites on the genome of PPV in 1992 (ref. # 1), but without giving a description or exact characterization of these sites. Also, no description of a successful application of PPV as vectors has yet been made.

In our own investigations into the sequence analysis of the HindIII fragment I of PPV strain D1701, an ORF with amino acid homology (36.1 to 38.3% identity, 52.8 to 58.6% similarity, GCG, Wisconsin Package 8.1, eg program Pikup ) to the vascular endothelial growth factor (VEGF) of various mammalian species (eg mouse, rat, guinea pig, bovine, human). Seq. ID No: 1 shows the nucleotide sequence of the gene in D1701, Seq. ID No: 15 is the amino acid sequence of the corresponding protein of D1701. Recently, a homologous gene has also been described in the PPV strains NZ2 and NZ7 (ref. # 6), but its function is not known. A corresponding gene is present in other poxviruses, e.g. Orthopoxviruses, not known. Later in the text, this gene is called the VEGF gene.

Our sequence analysis of the HindIII fragment I of D1701 led to the identification of another ORF that has homology to protein kinase genes of orthopox viruses and is known as Vaccinia as F10L. The identity to the vaccinia F10L gene is 51%, the similarity 70%. In the further course of the application, this gene is called the PK gene. Seq. ID No: 2, No: 9, No: 13 shows versions of the nucleotide sequence of the gene in D1701, Seq. ID No: 14 the amino acid sequence of the corresponding protein of D1701.

An ORF was also found to overlap the 3 'end of the PK gene and the 5' end of the VEGF gene. Homology studies showed low identity (28%) and low similarity (51%) to the F9L gene in vaccinia. Seq. ID No: 5, No: 10, shows versions of the nucleotide sequence of the gene at D1701. Later in the text, this gene is called the F9L gene.

Within the ITR region, another ORF was identified, which is called ORF3 because of its similarity to a gene in PPV NZ2 (identity 76%, similarity 83%). Seq. ID No: 4 shows the nucleotide sequence of the gene at D1701. In the further course of the text, this gene is called ORF3 gene.

1. 1. Rekombinant hergestellte PPV mit Insertionen und/oder Deletionen, enthaltend ein Fremdantigen, welches einen erregerspezifischen Schutz induzieren kann.
2. 2. Rekombinant hergestellte PPV mit Insertionen und/oder Deletionen, enthaltend ein Zytokin.
3. 3. Rekombinant hergestellte PPV gemäss 1 oder 2, enthaltend Insertionen und/oder Deletionen in Genomabschnitten, die nicht für die Virusvermehrung notwending sind.
4. 4. Rekombinant hergestellte PPV gemäss 1 oder 2, enthaltend Insertionen und/oder Deletionen in Genomabschnitten, die für die Virusvermehrung notwending sind.

The subject of the present invention are:

1. 1. Recombinantly produced PPV with insertions and / or deletions, containing a foreign antigen, which can induce a pathogen-specific protection.
2. 2. Recombinantly produced PPV with insertions and / or deletions containing a cytokine.
3. 3. Recombinantly produced PPV according to 1 or 2, containing insertions and / or deletions in genome sections, which are not necessary for the virus propagation.
4. 4. Recombinantly produced PPV according to 1 or 2, containing insertions and / or deletions in genome sections necessary for virus replication.

• Attenuierung ist ein
  Prozeß bei dem die PPV durch Veränderung in ihrem Genom vermindert- oder nicht-pathogen bzw. -virulent für Tiere oder Menschen geworden sind.
• Deletionen sind
  fehlende DNA-Teilstücke aus dem Genom von PPV.
• Deletionsplasmide sind
  Plasmide, die neben der Plasmid-DNA PPV-Genomabschnitte tragen, denen Teilstücke entfernt wurden.
• Zur Virusvermehrung notwendige (essentielle) Genomabschnitte sind
  Teile des Gesamt-Genoms von PPV, die zur in vitro-Vermehrung von PPV, d.h. zur Bildung von infektiösen Virusnachkommen unverzichtbar sind.
  Eine Störung von Genen, die für die Virusvermehrung essentiell sind, führt zu einer Unterbrechnung in der Virusvermehrung. Entfernt man beispielsweise Teile eines dieser Gene oder das gesamte Gen, bricht die Virusreplikation an einer bestimmten Stelle im Vermehrungszyklus des Virus ab. Infektionen oder Behandlungen mit solchen Mutanten führen nicht zu einer Freisetzung infektiöser Nachkommen aus dem Tier. Ersetzt man Teile eines oder ein gesamtes essentielles Gene(s) durch Fremd-DNA oder inseriert Fremd-DNA in essentielle Gene, können Vektorvakzinen konstruiert werden, die im Impfling nicht vermehrungsfähig sind und somit nicht als infektiöse Erreger ausgeschieden werden.
• Zur Virusvermehrung nicht-notwendige (nicht-essentielle) Genomabschnitte sind
  Teile des Gesamt-Genoms von PPV, die zur in vitro-Vermehrung von PPV, d.h. zur Bildung von infektiösen Virusnachkommen, verzichtbar sind.
• Fremde DNA-Elemente (Fremd-DNA) sind
  DNA-Teilstücke, z.B. Fremd-Gene oder Nukleotidsequenzen, die in dem erfindungsgemäß eingesetzten PPV ursprünglich nicht vorhanden sind.
  Fremd-DNA wird aus folgenden Gründen in das PPV inseriert:
  1. 1. zur Expression der Fremd-DNA
  2. 2. zur Inaktivierung von Funktionen von DNA-Teilstücken des PPV
  3. 3. zur Markierung des PPV.
  
  In Abhängigkeit von diesen Gründen wird unterschiedliche Fremd-DNA inseriert. Soll gemäß (1) Fremd-DNA exprimiert werden, so wird die inserierte Fremd-DNA mindestens einen offenen Leserahmen tragen, der für ein oder mehrere gewünschte Fremdprotein(e) kodiert. Gegebenenfalls enthält die Fremd-DNA zusätzlich eigene oder fremde Regulatorsequenzen. Die Kapazität zur Aufnahme von Fremd-DNA kann durch Setzen von Deletionen im Genom des Virus vergrößert werden. Im allgemeinen liegt die Länge zwischen 1-Nukleotid und 35.000, bevorzugt zwischen 100 und etwa 15.000 Nukleotiden.
  Als Beispiele seien genannt, Gene oder Genteile von Viren, wie beispielsweise
  • Herpesvirus suid 1,
  • Equine Herpesviren
  • Bovine Herpesviren
  • Maul- und Klauenseuche-Virus,
  • Bovines Respiratorisches Syncytial Virus,
  • Parainfluenzavirus 3 des Rindes,
  • Influenzavirus,
  • Calicivirus
  • Flaviviren, z.B. Bovines Virus Diarrhoe Virus oder Virus der klassischen Schweinepest
  oder von Bakterien, wie beispielsweise
  • Pasteurella spec.;
  • Salmonella spec.,
  • Actinobacillus spec.,
  • Chlamydia spec.,
  oder von Parasiten, wie beispielsweise
  • Toxoplasma,
  • Dirofilaria,
  • Echinococcus.
  
  Soll gemäß (2) Fremd-DNA inseriert werden, reicht für die Unterbrechung der Vektor-Virus-DNA-Sequenz prinzipiell die Insertion eines geeigneten Fremdnukleotids. Die maximale Länge der zur Inaktivierung inserierten Fremd-DNA richtet sich nach der Aufnahmekapazität des Vektor-Virus für Fremd-DNA. Im allgemeinen ist die Länge der Fremd-DNA zwischen 1 Nukleotid und 35.000 Nukleotiden, bevorzugt zwischen 100 und 15.000 Nukleotiden, besonders bevorzugt zwischen 3 bis 100 Nukleotiden.
  Sollen gemäß (3) DNA-Sequenzen zur Markierung inseriert werden, richtet sich ihre Länge nach der Nachweismethode zur Identifizierung des markierten Virus. Im allgemeinen ist die Länge der Fremd-DNA zwischen 1 und 25.000 Nukleotiden, bevorzugt zwischen 20 Nukleotiden und 15.000 Nukleotiden, besonders bevorzugt zwischen 5 bis 100 Nukleotiden.
• Genbank
  ist die Gesamtheit von in vermehrungsfähigen Vektoren enthaltenen Fragmente eines Genoms. Sie wird durch Fragmentierung des Genoms und Insertion aller, einzelner oder eines Großteils der Fragmente in vermehrungsfähige Vektoren, wie z.B. Plasmide, erhalten.
• Genomfragment ist
  ein Teilstück eines Genoms, das isoliert vorliegen oder in einen vermehrungsfähigen Vektor inseriert sein kann.
• Inaktivierung durch Insertion bedeutet,
  daß die inserierte Fremd-DNA die Expression oder Funktion PPV-eigener Genom-Sequenzen verhindert.
• Insertionen sind
  DNA-Teilstücke, die zusätzlich in das Genom von PPV eingebaut wurden. Die Länge der DNA-Teilstücke kann je nach Grund der Insertion zwischen 1 Nukleotid und mehreren tausend Nukleotiden liegen (siehe auch Definition für "Fremd-DNA").
• Insertionsplasmide sind
  Plasmide, insbesondere bakterielle Plasmide, die die zu inserierende Fremd-DNA, flankiert von DNA-Sequenzen des PPV, beinhalten.
• Insertionsstellen sind
  Stellen in einem Virus-Genom, die zur Aufnahme von Fremd-DNA geeignet sind.
• Klonierung heißt,
  daß die genomische DNA von PPV isoliert und fragmentiert wird. Die Fragmente oder eine Auswahl der Fragmente werden in gängige DNA-Vektoren (bakterielle Plasmide oder Phagenvektoren oder eukaryontische Vektoren) inseriert.
  Eine Auswahl an Methoden zur Herstellung und Klonierung von DNA-Fragmenten gibt Lit. #11. Die DNA-Vektoren mit den PPV-DNA-Fragmenten als Inserts dienen z.B. der Herstellung von identischen Kopien der ursprünglich isolierten DNA-Fragmente von PPV.
• Markierung durch Insertion bedeutet,
  daß die inserierte Fremd-DNA die spätere Identifizierung des veränderten PPV ermöglicht.
• Unter ORF (Open Reading Frame) versteht man eine Abfolge von Nukleotiden auf DNA-Ebene, die die Aminosäuresequenz eines potentiellen Proteins definiert. Sie besteht aus einer durch die Größe des von ihr definierten Proteins bestimmten Anzahl von Nukleotid-Tripletts, die 5'-seitig durch ein Startcodon (ATG) und 3'-seitig durch ein Stopcodon (TAG, TGA, TAA) begrenzt wird.
• Regulatorsequenzen sind
  DNA-Sequenzen, die die Expression von Genen beeinflussen. Solche sind bekannt aus Lit. #15.
  Bevorzugt sei genannt der VEGF-Promotor, wie er in Sequenz Protokoll ID No: 6 beschrieben ist.
• Rekombinante PPV sind
  PPV mit Insertionen und/oder Deletionen in ihrem Genom. Die Insertionen und Deletionen wurden hierbei über molekularbiologische Methoden hergestellt.
• Repetitive (DNA)-Sequenzen sind
  identische Nukleotid-Sequenzen, die im PPV-Genom direkt nacheinander oder verstreut an unterschiedlichen Stellen vorkommen.
• Vektor-Virus ist
  ein PPV, das zur Insertion von Fremd-DNA geeignet ist und die inserierte Fremd-DNA in seinem Genom in infizierte Zellen oder Organismen transportieren kann und gegebenenfalls die Expression der Fremd-DNA ermöglicht.

The terms listed above have the following meaning:

• Attenuation is one
  Process in which the PPV have become degraded or non-pathogenic or virulent to animals or humans by alteration in their genome.
• Deletions are
  missing DNA fragments from the genome of PPV.
• Deletion plasmids are
  Plasmids that carry PPV genome sections in addition to the plasmid DNA that have been removed.
• Necessary for virus replication (essential) genome sections are
  Portions of the total genome of PPV indispensable for the in vitro propagation of PPV, that is, for the generation of infectious progeny virus.
  A disruption of genes that are essential for virus replication leads to an interruption in the virus multiplication. If, for example, parts of one of these genes or the entire gene are removed, virus replication breaks down at a certain point in the reproductive cycle of the virus. Infections or treatments with such mutants do not result in the release of infectious offspring from the animal. Replacing parts of or an entire essential gene (s) by foreign DNA or inserting foreign DNA into essential genes, vector vaccines can be constructed that are not replicable in the vaccine and thus are not excreted as infectious agents.
• For virus replication, non-essential (nonessential) genome segments are
  Parts of the total genome of PPV, which are dispensable for the in vitro propagation of PPV, ie for the formation of infectious virus progeny.
• Foreign DNA elements (foreign DNA) are
  DNA fragments, eg foreign genes or nucleotide sequences, which are not originally present in the PPV used according to the invention.
  Foreign DNA is inserted into the PPV for the following reasons:
  1. 1. to the expression of the foreign DNA
  2. 2. Inactivation of functions of DNA fragments of the PPV
  3. 3. to mark the PPV.
  
  Depending on these reasons, different foreign DNA is inserted. If foreign DNA is to be expressed according to (1), the inserted foreign DNA will carry at least one open reading frame which codes for one or more desired foreign protein (s). Optionally, the foreign DNA additionally contains its own or foreign regulatory sequences. The capacity for uptake of foreign DNA can be increased by setting deletions in the genome of the virus. In general, the length is between 1 nucleotide and 35,000, preferably between 100 and about 15,000 nucleotides.
  Examples include genes or genetic parts of viruses, such as
  • Herpesvirus suid 1,
  • Equine herpesviruses
  • Bovine herpesviruses
  • Foot-and-mouth disease virus,
  • Bovine Respiratory Syncytial Virus,
  • Parainfluenza virus 3 of bovine,
  • Influenza virus,
  • calicivirus
  • Flaviviruses, eg Bovine virus diarrhea virus or classical swine fever virus
  or of bacteria, such as
  • Pasteurella spec .;
  • Salmonella spec.,
  • Actinobacillus spec.,
  • Chlamydia spec.,
  or of parasites, such as
  • Toxoplasma,
  • Dirofilaria,
  • Echinococcus.
  
  If according to (2) foreign DNA is inserted, the insertion of a suitable foreign nucleotide is sufficient in principle for the interruption of the vector virus DNA sequence. The maximum length of the foreign DNA inserted for inactivation depends on the uptake capacity of the vector virus for foreign DNA. In general, the length of the foreign DNA is between 1 nucleotide and 35,000 nucleotides, preferably between 100 and 15,000 nucleotides, more preferably between 3 to 100 nucleotides.
  If, according to (3), DNA sequences are to be inserted for labeling, their length depends on the detection method for identification of the labeled virus. In general, the length of the foreign DNA is between 1 and 25,000 nucleotides, preferably between 20 nucleotides and 15,000 nucleotides, more preferably between 5 to 100 nucleotides.
• Genbank
  is the set of fragments of a genome contained in replicable vectors. It is obtained by fragmenting the genome and inserting all, a single or a majority of the fragments into replicable vectors, such as plasmids.
• Genome fragment is
  a portion of a genome that may be isolated or inserted into a replicable vector.
• Inactivation by insertion means
  that the inserted foreign DNA prevents the expression or function of PPV's own genome sequences.
• Insertions are
  DNA fragments additionally incorporated into the genome of PPV. The length of the DNA fragments can be between 1 nucleotide and several thousand nucleotides, depending on the reason for the insertion (see also definition for "foreign DNA").
• Insertion plasmids are
  Plasmids, in particular bacterial plasmids, which contain the foreign DNA to be inserted, flanked by DNA sequences of the PPV.
• Insertion sites are
  Sites in a virus genome that are suitable for receiving foreign DNA.
• Cloning means
  that the genomic DNA of PPV is isolated and fragmented. The fragments or a selection of the fragments are inserted into common DNA vectors (bacterial plasmids or phage vectors or eukaryotic vectors).
  For a selection of methods for preparing and cloning DNA fragments, see Ref. # 11. The DNA vectors with the PPV DNA fragments as inserts serve, for example, to produce identical copies of the originally isolated DNA fragments of PPV.
• Marking by insertion means
  that the inserted foreign DNA allows later identification of the altered PPV.
• ORF (Open Reading Frame) is a sequence of DNA-level nucleotides that defines the amino acid sequence of a potential protein. It consists of a number of nucleotide triplets determined by the size of the protein defined by it, which is bounded on the 5 'side by a start codon (ATG) and on the 3' side by a stop codon (TAG, TGA, TAA).
• Regulator sequences are
  DNA sequences that influence the expression of genes. Such are known from Ref. # 15.
  Preference should be given to the VEGF promoter as described in Sequence Protocol ID No: 6.
• Recombinant PPV are
  PPV with insertions and / or deletions in their genome. The insertions and deletions were produced by molecular biological methods.
• Repetitive (DNA) sequences are
  identical nucleotide sequences that occur directly in the PPV genome or scattered at different sites.
• Vector virus is
  a PPV which is suitable for insertion of foreign DNA and which can transport the inserted foreign DNA in its genome into infected cells or organisms and optionally allows expression of the foreign DNA.

1.

Auswahl eines geeigneten PPV-Stammes

2.

Identifizierung von Genomabschnitten mit Insertionsstellen in dem Genom des PPV

2.a

Identifizierung von PPV-Genomabschnitten mit Insertionsstellen in Genen, die nicht-essentiell für die Virusvermehrung sind,

2.b

Identifizierung von PPV Genomabschnitten mit Insertionsstellen in Genen, die essentiell für die Virusvermehrung sind,

2.c

Identifizierung von Genomabschnitten mit Insertionsstellen in Bereichen außerhalb von Genen in dem Genom des PPV und/oder in Genduplikationen

2.d

Weitere Methoden zur Identifizierung von Genomabschnitten mit Insertionsstellen

2.e

Anforderungen an eine Insertionsstelle

2.1

Identifizierung von Insertionsstellen

2.1.1

Reinigung des Genoms von PPV

2.1.2

Klonierung der Genomfragmente, Etablierung einer Genbank

2.1.3

Sequenzierung zur Identifizierung von Genen oder Genomabschnitten außerhalb von Genen

2.1.4

Auswahl der Klone mit PPV-Genomfragmenten zur Weiterverarbeitung

2.2

ITR-Region, VEGF-, PK-Gen, Gen kodierend für das 10 kDa-Protein und der Bereich zwischen dem PK- und dem HDIR-Gen als Insertionsstellen

2.2.1

Klonierung des VEGF-Gens

2.2.2

Klonierung des Proteinkinase-Gens

2.2.3

Klonierung des Gen-Bereichs der für das 10kDa-Protein kodiert

2.2.4

Klonierung der ITR-Region (Inverted-Terminal-Repeat-Region) bzw. des Genomabschnittes, der zwischen dem PK-Gen und dem HD1R-Gen liegt

3.

Konstruktion von Insertionsplasmiden oder von Deletionsplasmiden, die die zu inserierende Fremd-DNA enthalten,

3.1

Identifizierung oder Herstellung von nur einmal vorkommenden, d.h. singulären Restriktionsenzymerkennungsstellen ("unique restriction sites") in den klonierten Genomfragmenten und Insertion von Fremd-DNA

3.2

Deletion von Genomsequenzen in den klonierten Genomfragmenten und Insertion von Fremd-DNA

3.3

Eine Kombination von #3.1 und #3.2

4.

Konstruktion eines rekombinanten PPV

The preparation of the PPV according to the invention is described as follows:

1.

Selection of a suitable PPV strain

Second

Identification of genomic regions with insertion sites in the genome of the PPV

2.a

Identification of PPV genomic regions with insertion sites in genes that are non-essential for virus replication,

2 B

Identification of PPV genome segments with insertion sites in genes essential for virus replication

2.c

Identification of genomic regions with insertion sites in out-of-gene regions in the genome of the PPV and / or in gene duplications

2.d

Further methods for the identification of genome sections with insertion sites

2.e

Requirements for an insertion site

2.1

Identification of insertion sites

2.1.1

Purification of the genome of PPV

2.1.2

Cloning of genomic fragments, establishment of a gene bank

2.1.3

Sequencing for the identification of genes or genome segments outside of genes

2.1.4

Selection of clones with PPV genome fragments for further processing

2.2

ITR region, VEGF, PK gene, gene coding for the 10 kDa protein and the region between the PK and the HDIR gene as insertion sites

2.2.1

Cloning of the VEGF gene

2.2.2

Cloning of the protein kinase gene

2.2.3

Cloning the gene region encoding the 10kDa protein

2.2.4

Cloning of the ITR region (inverted terminal repeat region) or of the genome segment which lies between the PK gene and the HD1R gene

Third

Construction of insertion plasmids or of deletion plasmids containing the foreign DNA to be inserted,

3.1

Identification or production of unique, ie unique restriction restriction recognition sites in the cloned genomic fragments and insertion of foreign DNA

3.2

Deletion of genome sequences in the cloned genomic fragments and insertion of foreign DNA

3.3

A combination of # 3.1 and # 3.2

4th

Construction of a recombinant PPV

1. Selection of a suitable PPV strain

Basically, all PPV species are suitable for carrying out the present invention. Preference is given to virus strains which can be multiplied to titer> 10 ⁵ PFU (plaque forming unit) / ml in a tissue culture and can be purified from the medium of the infected cells as an extracellular, infectious virus. Preference is given to the genus of PPV the species PPV ovis (Orf viruses).

Particular preference is given to the PPV ovis strain D1701, deposited under the Budapest Treaty on 28.04.1988 at Institut Pasteur, CNCM under Reg.No. CNCM I-751, as well as its variants and mutants.

The multiplication of the viruses takes place in a customary manner in tissue cultures of animal cells, such as mammalian cells, for example in ovine cells, bovine cells, preferably in bovine cells such as the permanent bovine kidney cell BK-K1-3A (or its progeny) or monkey cells such as the permanent monkey kidney cells MA104 or Vero (or their progeny).

Propagation is carried out in a manner known per se in stationary, roller or carrier cultures in the form of closed cell aggregates or in suspension cultures.

The cells and cell turfs used for the multiplication of the viruses are increased in the usual way almost to confluence or up to optimal cell density. The cells are infected with virus dilutions that correspond to an MOI (= multiplicity of infection, corresponding to infectious virus particles per cell).

The multiplication of the viruses takes place with or without the addition of animal serums. In the event that serum is used, this is added to the propagation medium in a concentration of 1-30 vol .-%, preferably 1-10 vol .-%.

Infection and virus multiplication takes place at temperatures between room temperature and 40 ° C, preferably between 32 and 39 ° C, more preferably at 37 ° C for several days, preferably until complete destruction of the infected cells. In the virus harvest, cell-bound virus can be mechanically or by ultrasound or mild enzymatic proteolysis e.g. be additionally released with trypsin.

The virus-containing medium of the infected cells can then be further worked up, e.g. by removing the cell debris by filtration with pore sizes of e.g. 0.2-0.45 μm and / or low speed centrifugation.

Filtrate or centrifugation supernatant can be used for virus replenishment and purification. For this purpose, the filtrate or supernatant of a high-speed centrifugation are subjected to sedimentation of the virus particles. Optionally, further purification steps can be connected by, for example, centrifugation in a density gradient.

2. Identification of genomic sections with insertion sites in the genome of the PPV

1. a. in Gene, die nicht-essentiell für die Virusvermehrung in vitro und/oder in vivo sind, .
2. b. in Gene, die essentiell für die Virusvermehrung sind und/oder
3. c. in Bereichen ohne Genfunktion.

Different regions of the genome of the PPV can serve as insertion sites for insertion of foreign DNA. Foreign DNA can be inserted,

1. a. in genes that are non-essential for viral replication in vitro and / or in vivo,.
2. b. in genes that are essential for virus replication and / or
3. c. in areas without gene function.

2.a Identification of genomic regions with insertion sites in genes that are non-essential for virus propagation in the genome of the PPV

• i. Viral genes that are non-essential for virus propagation are found, for example, by comparative studies with representatives of various PPV species. Genes that are not found in one or more isolates or strains of a PPV species but are found in other isolates or strains are potentially non-essential.
• ii. Genes that are non-essential for virus replication can also be identified by an alternative route. Following sequencing of genomic fragments of PPV that may be present, for example, as cloned fragments, these DNA sequences are screened for possible "open reading frames" (ORFs). If an ORF is found, its function as a gene is verified via the detection of transcription and / or translation. To determine if the gene found is non-essential for virus replication, the gene is removed from the genome of the PPV by molecular genetic methods, partially disrupted or disrupted by introduced mutation (s), and the resulting virus assayed for replicative capacity. If the virus can also replicate without the existence of the manipulated gene, this is a non-essential gene.

Examples of identified insertion sites

1. i. An example of a non-essential gene of PPV is the VEGF gene of the PPV ovis, which acts as an inducible site for foreign DNA. This gene is found in the strains studied by Parapoxvirus ovis (NZ-2, NZ-7 and D1701 (Ref. # 6)). In some PPV strains, for example, members of PPV bovis 1, this VEGF gene is not detected. The identification of the region with the VEGF gene on the genome of PPV can be carried out using the DNA sequence shown in Sequence Protocol ID No: 1. By conventional molecular biology techniques, the gene can be found by hybridization experiments, genome sequence analyzes, and / or polymerase chain reactions on the genome of a PPV.
2. ii. Another example of a potentially non-essential gene of PPV is the gene for the 10kDa protein of PPV. This gene is found in strains of Parapoxvirus ovis (NZ-2, NZ-7 and D1701). The identification of the region for the 10kDa protein gene on the genome of PPV can be found on the genome of a PPV using standard molecular biology techniques, such as polymerase chain reaction (PCR). Ref. # 8 shows the DNA sequence of the 10kDa protein gene. The primers which can be used for a PCR are mentioned, for example, in reference # 8. The DNA sequence of the 10kDa-specific PCR product of D1701 shows Sequence Listing ID No: 11.

For the insertion of foreign DNA, the non-essential gene may be partially or completely deleted from the genome of the PPV. However, it is also possible to insert foreign DNA into the non-essential gene without removing portions of the PPV gene. For example, restriction enzyme recognition sites can serve as insertion sites.

2.b Identification of PPV genomic regions with insertion sites in genes that are essential on the genome of the PPV

1. i. The identification of essential genes can be accomplished by sequencing viral genomic fragments present, for example, as cloned fragments, and identifying possible ORFs.
   If an ORF is found, its function as a gene is verified via the detection of transcription and / or translation. To determine if the gene found is essential for virus propagation, the gene is disrupted by molecular genetic methods in the genome of the PPV, for example, by removal of parts or the whole gene, or by insertion of foreign DNA, and the resulting virus is tested for proliferation. If the viral mutant obtained can not replicate, it is likely to be an essential gene.

Proof is the exclusive growth of the virus mutant in complementing cell lines.

Examples of insertion sites

As an example, the protein kinase gene (PK gene) of PPV D 1701 called. The PK gene is expressed late in the reproductive cycle of the virus. The identification of the PK gene on the genome of PPV can be done using the versions of the DNA sequence shown in Sequence Listing ID No: 2, No: 9 or No: 13. By the usual methods of molecular biology, the region of the gene can be found, for example, by hybridization experiments, genome sequence analyzes and / or polymerase chain reactions on the genome of a PPV.

For the insertion of foreign DNA, the essential gene can be partially or completely removed from the genome of the PPV. However, it is also possible to insert the foreign DNA into the essential gene without removing areas from the PPV gene.

2.c Identification of genomic regions with insertion sites in non-gene regions on the genome of the PPV and / or in gene duplications

Genome segments which do not code for functional gene products and have no essential regulatory functions (so-called intergenic sections) are in principle suitable as insertion sites for foreign DNA. Particularly suitable are areas with repetitive sequences, since changes in partial areas can be compensated by remaining Sequenzrepetitionen. This includes genes that occur in two or more copies, so-called gene duplications.

1. i. Die Identifizierung von Genomsequenzen, die nicht für Genprodukte kodieren, erfolgt über Sequenzanalysen des Genoms der PPV. Genombereiche, die weder nach Sequenzanalyse einen ORF noch virusspezifische Transkription zeigen und keine regulatorische Funktion aufweisen, stellen potentielle Insertionsstellen dar. Besonders die Schnittstellen für Restriktionsenzyme in diesen Bereichen repräsentieren potentielle Insertionsstellen. Zur Überprüfung, ob eine geeignete Insertionsstelle vorliegt, wird über bekannte molekularbiologische Methoden Fremd-DNA in die potentielle Insertionsstelle inseriert und die resultierende Virusmutante auf Lebensfähigkeit untersucht. Wenn die Virusmutante, die in der möglichen Insertionstelle Fremd-DNA trägt, vermehrungsfähig ist, handelt es sich bei der untersuchten Stelle um eine geeignete Insertionstelle.
2. ii. Die Identifizierung von repetitiven Sequenzen sowie Genduplikationen erfolgt beispielsweise über DNA-Hybridisierungsexperimente und/oder über Sequenzanalysen. Bei den Hybridisierungsexperimenten werden klonierte oder isolierte Genomfragmente eines PPV als Sonden für Hybridisierungen mit PPV-DNA-Fragmenten eingesetzt. Die Genomfragmente des PPV, die mit mehr als einem Fragment des Gesamtgenoms des PPV hybridisieren, enthalten eine oder mehrere repetitive Sequenzen. Um die repetitive Sequenz oder duplizierte Genombereiche auf dem Genom-Fragment genau zu lokalisieren, wird die Nukleotidsequenz bestimmt. Um festzustellen, ob eine potentielle Insertionsstelle eine geeignete Insertionsstelle im Gesamtgenom des PPV ist, muß Fremd-DNA in eine repetitive Sequenz oder in eine Kopie der Genduplikation inseriert werden und das PPV-Genom-Fragment mit dem Insert in das Virusgenom eingebaut werden. Anschließend wird das Fremd-DNA enthaltende rekombinante Virus auf Vermehrungsfähigkeit geprüft. Vermehrt sich das rekombinante Virus, ist die identifizierte Erkennungsstelle als Insertionsstelle geeignet.

Genes in the ITR region or in duplicated sections of the PPV genome exist in two copies in the viral genome. After removal or alteration of a copy of such a gene and insertion of foreign DNA, stable PPV recombinants can be obtained, even though the altered gene should be important for virus propagation. A second, unaltered gene copy might be sufficient for gene function.

1. i. The identification of genome sequences which do not code for gene products is carried out via sequence analyzes of the genome of the PPV. Genome regions which show neither ORF nor virus-specific transcription after sequence analysis and have no regulatory function represent potential insertion sites. In particular, the restriction enzyme sites in these areas represent potential insertion sites. To check whether a suitable insertion site is present, foreign DNA is inserted into the potential insertion site via known molecular-biological methods and the resulting virus mutant is examined for viability. If the virus mutant carrying foreign DNA in the possible insertion site is capable of reproduction, the site of interest is an appropriate insertion site.
2. ii. The identification of repetitive sequences as well as gene duplications takes place, for example, via DNA hybridization experiments and / or via sequence analyzes. In hybridization experiments, cloned or isolated genomic fragments of a PPV are used as probes for hybridization with PPV DNA fragments. The genomic fragments of the PPV that hybridize to more than one fragment of the total genome of the PPV contain one or more repetitive sequences. To the repetitive sequence or duplicated To accurately locate genomic regions on the genome fragment, the nucleotide sequence is determined. To determine if a potential insertion site is a suitable insertion site in the overall genome of the PPV, foreign DNA must be inserted into a repetitive sequence or into a copy of the gene duplication and the PPV genome fragment with the insert inserted into the viral genome. Subsequently, the foreign DNA-containing recombinant virus is tested for proliferative capacity. If the recombinant virus proliferates, the identified recognition site is suitable as an insertion site.

Examples of insertion sites

1. i. Examples of intergenic regions include the genome section between the gene for the protein kinase and the HDIR gene (sequence protocol ID No: 7).
2. ii. An example of a repetitive sequence is called the ITR region (Sequence Protocol ID No: 4).
3. iii. As an example of a gene duplication in the PPV strain D1701, the potential gene "ORF 3" (in the ITR region) and the VEGF gene may be mentioned. Hybridization studies demonstrated that in the present strain D1701, an area containing the VEGF gene was duplicated and translocated to the other end of the viral genome so that there are two copies of VEGF gene.
   Based on the sequences in sequence protocol ID No: 4 (ITR sequence with gene "ORF3") and ID No: 7 (region between PK and HDIR gene), the corresponding genome regions in other PPVs can be analyzed by conventional methods of molecular biology, for example hybridization experiments , Genome sequence analyzes and / or polymerase chain reactions can be found.

2.d Further methods for the identification of insertion sites

In general, possible insertion sites on the genome of PPV are also found via modifications of the viral genome sequences. Genomic sites where nucleotide exchanges, deletions and / or insertions or combinations thereof do not block viral replication represent potential sites of insertion. To test whether a potential site of insertion is a suitable insertion site, foreign DNA is introduced into the potential by known molecular biology techniques Insertion site inserted and examined the resulting virus mutant for viability. If the virus recombinant is capable of replication, the site of interest is an appropriate insertion site.

2.1 Identification of insertion sites 2.1.1 Purification of the genome of PPV

For the molecular genetic cloning of insertion sites of PPV, the genome of the PPV is first purified. Starting from the virus prepared according to 1 (above), the genome is isolated and purified. The extraction of native viral DNA is preferably carried out by treating the purified virions with aqueous solutions of detergents and proteases.

Detergents include anionic, cationic, amphoteric, nonionic detergents. Preferably, ionic detergents are used. Particularly preferred are sodium dodecyl sulfate, sodium lauryl sulfate.

As proteases may be mentioned all proteases which operate in the presence of detergent, e.g. Proteinase K and pronase. Preference is given to proteinase K.

Detergents are used in concentrations of 0.1-10 vol .-%, preferably 0.5-3 vol .-%.

Proteases are used in concentrations of 0.01-10 mg / ml virus lysate, preferably 0.05-0.5 mg / ml virus lysate.

It is preferred to work in aqueous buffered solution in the presence of DNase inhibitors. As buffer substances may be mentioned: salts of weak acids with strong bases such as e.g. Tris (hydroxymethylaminomethane), salts of strong acids with weak bases, e.g. primary phosphates or mixtures thereof.

The following buffer system may preferably be mentioned: tris (hydroxymethylaminomethane).

The buffer substances or buffer systems are used in concentrations which ensure pH values at which the DNA does not denature. Preference is given to pH values of 5-9, particularly preferably 6-8.5, completely particularly preferred 7-8, in particular work in the neutral range is called.

DNase inhibitors are e.g. Ethylenediaminetetraacetic acid in concentrations of 0.1-10 mM (millimoles), preferably about 1 mM.

Subsequently, the lipophilic components of the virus lysate are extracted. The extractants used are solvents such as phenol, chloroform, isoamyl alcohol or mixtures thereof. Preferably, a mixture of phenol and chloroform / isoamyl alcohol is first used, wherein the extraction takes place in one or more stages.

Other methods of isolating the viral DNA are e.g. Centrifugation of a viral lysate in a CsCl density gradient or in gel electrophoresis (see Ref. # 14).

Extraction of nucleic acids is described in Ref. # 13.

The DNA so extracted is preferably extracted from the aqueous solution with e.g. Alcohol, preferably with ethanol or isopropanol and with the addition of monovalent salts such. Alkali chlorides or acetates, preferably lithium chloride, sodium chloride or sodium acetate, potassium acetate, precipitated (see loc cit.).

2.1.2 Cloning of Genome Fragments

The purified viral DNA is now used to produce DNA fragments. It is treated for this purpose, for example, with restriction enzymes. Suitable restriction enzymes are, for example, Eco RI, BamHI Hin dIII, KpnI. Alternatively, genome fragments can be synthesized by polymerase chain reaction (PCR). For this purpose, primers are selected from already known sequence segments of the virus genome and synthesized by means of, for example, Taq or Pfu polymerase of the primer pair-limited genome section in vitro.

The DNA fragments resulting from restriction digestion or PCR can be cloned into vector systems according to the methods described in (Sambrock 89) become. For this purpose, for example, depending on the size of the particular DNA fragment to be cloned, plasmid vectors, lambda phage vectors or cosmid vectors are suitable.

2.1.3 Sequencing. Identification and characterization of genes. Verification of their expression

Genomic fragments cloned into vectors are first analyzed by sequencing. The inserted DNA fragments are mapped by means of various restriction enzymes and suitable subfragments are cloned into plasmid vectors. The sequencing reaction is carried out, for example, using the T7 sequencing kit from Pharmacia according to the manufacturer. The double-stranded plasmid DNA required for this purpose is preferably prepared by the PEG method (Hattori and Sakaki 1985). By means of computer analysis (CGC, see above), "open reading frames (ORF)" present on the genome fragments are identified. Insights into the respective function of the identified ORFs can be obtained by comparing their sequence with other gene sequences of known function present in a database. Functionally, the identified ORFs are characterized by detection of their respective corresponding transcripts in virus-infected cells. For this purpose, the total RNA of virus-infected cells is isolated, for example, by the AGPC method (Chomczynski and Sacchi, (20) 1987). By Northern blot analysis or primer extension and RNA protection experiments, the specific transcripts can be identified together with their 5 'and 3' ends. Alternatively, it is possible to express the virus protein encoded by an identified ORF in vitro, to obtain antisera by means of the expression product and to detect the expression of the ORF.

To determine whether the gene found is non-essential for virus replication, the gene can be destroyed by gene disruption or gene deletion. In this case, either the entire gene or parts thereof are removed from the PPV genome or the reading frame of the gene is interrupted by the insertion of foreign gene sequences. The resulting virus is tested for reproductive ability. Can the virus even without the Replicating the existence of the destroyed gene, this is a non-essential gene.

2.1.4 Selection of Clones with PPV Genome Fragments

1. i. Sollen rekombinante PPV hergestellt werden, die trotz der Insertion und/oder Deletion replikationsfähig sind, werden klonierte virale Genomfragmente weiterverarbeitet, die Gene oder Genombereiche außerhalb von Genen enthalten, die nicht-essentiell für die Virusvermehrung sind oder Genduplikationen enthalten.
   Die Prüfung, ob es sich bei dem vorliegenden Gen oder Genombereich um eine nicht-essentielle Region des Virusgenoms oder um eine Genduplikation handelt, wird über Virusmutanten bestimmt. Hierzu wird das untersuchte Gen bzw. der untersuchte Genombereich im PPV durch molekularbiologische Methoden inaktiviert, beispielsweise durch teilweise oder vollständige Deletion des fraglichen Bereiches, und die Vermehrungsfähigkeit der Virusmutante untersucht. Kann die Virusmutante sich trotz der Inaktivierung des fraglichen Gens oder Genombereiches vermehren, handelt es sich bei dem untersuchten Gen oder Genombereich um eine nicht-essentielle Region.
   Bevorzugt werden klonierte Genomfragmente des PPV, die die nicht-essentiellen Gene vollständig enthalten. Darüber hinaus sollten an beiden Enden der Gene oder der Genombereiche die flankierenden viralen Genombereiche ebenfalls enthalten sein. Die Länge der flankierenden Bereiche sollte mehr als 100 Basenpaare betragen. Liegen solche Genomklone nicht vor, können sie aus bestehenden Genomklonen mit molekularbiologischen Methoden hergestellt werden. Enhalten die klonierten Genomfragmente zusätzliche Genombereiche, die für die vorliegende Herstellung nicht benötigt werden, können diese durch Subklonierungen entfernt werden.
2. ii. Sollen rekombinante PPV hergestellt werden, die aufgrund der Insertion und/oder Deletion die Fähigkeit zur Bildung von infektiösen Nachkommen verloren haben, werden klonierte virale Genomfragmente weiterverarbeitet, die Gene oder Genombereiche außerhalb von Genen enthalten, die essentiell für die Virusvermehrung sind.
   Die Prüfung, ob es sich bei dem vorliegenden Gen oder Genombereich um eine essentielle Region des Virusgenoms handelt, kann über Virusmutanten bestimmt werden. Hierzu wird das untersuchte Gen bzw. der untersuchte Genombereich im PPV durch molekularbiologische Methoden inaktiviert, beispielsweise durch teilweise oder vollständige Deletion des fraglichen Bereiches, und die Vermehrungsfähigkeit der Virusmutante untersucht. Kann die Virusmutante sich aufgrund der Inaktivierung des fraglichen Gens oder Genombereiches nicht mehr vermehren, handelt es sich bei dem untersuchten Gen oder Genombereich um eine essentielle Region.
   Bevorzugt werden klonierte Genomfragmente des PPV, die die essentiellen Gene vollständig enthalten. Darüber hinaus sollten an beiden Enden der Gene oder der Genombereiche die flankierenden viralen Genombereiche ebenfalls enthalten sein. Die Länge der flankierenden Bereiche sollte mehr als 100 Basenpaare betragen. Liegen solche Genomklone nicht vor, können sie aus bestehenden Genomklonen mit molekularbiologischen Methoden hergestellt werden. Enhalten die klonierten Genomfragmente zusätzliche Genombereiche, die für die vorliegende Herstellung nicht benötigt werden, können diese durch Subklonierungen entfernt werden.

Which of the clones obtained above are used with genome fragments of the PPV depends on whether the recombinant PPV to be produced (i) should be able to replicate or (ii) be replication-defective.

1. i. If recombinant PPVs are to be produced which are capable of replication despite the insertion and / or deletion, cloned viral genome fragments are further processed which contain genes or genomic regions outside of genes which are non-essential for virus propagation or contain gene duplications.
   The test of whether the gene or genome region in question is a non-essential region of the virus genome or a gene duplication is determined by virus mutants. For this purpose, the investigated gene or the investigated genome region in the PPV is inactivated by molecular biological methods, for example by partial or complete deletion of the region in question, and examined the proliferation of the virus mutant. If the virus mutant can multiply despite the inactivation of the gene or genome region in question, the gene or genome region examined is a non-essential region.
   Preference is given to cloned genome fragments of the PPV which completely contain the nonessential genes. In addition, flanking viral genomic regions should also be included at both ends of the genes or genomic regions. The length of the flanking areas should be more than 100 base pairs. If such genome clones are not available, they can be prepared from existing genome clones using molecular biological methods. If the cloned genome fragments contain additional genome regions which are not suitable for the present preparation are needed, they can be removed by subcloning.
2. ii. If recombinant PPVs are to be produced which have lost the ability to form infectious offspring due to insertion and / or deletion, cloned viral genome fragments are further processed which contain genes or genomic regions outside of genes which are essential for virus replication.
   The test as to whether the gene or genome region in question is an essential region of the viral genome can be determined by virus mutants. For this purpose, the investigated gene or the investigated genome region in the PPV is inactivated by molecular biological methods, for example by partial or complete deletion of the region in question, and examined the proliferation of the virus mutant. If the virus mutant can no longer multiply due to the inactivation of the gene or genome region in question, the gene or genome region examined is an essential region.
   Preference is given to cloned genome fragments of the PPV that completely contain the essential genes. In addition, flanking viral genomic regions should also be included at both ends of the genes or genomic regions. The length of the flanking areas should be more than 100 base pairs. If such genome clones are not available, they can be prepared from existing genome clones using molecular biological methods. If the cloned genomic fragments contain additional genome regions which are not required for the present preparation, these can be removed by subcloning.

2.2 ITR region VEGF gene, PK gene, gene encoding the 10kDa protein and the region between the PK gene and the HD1R gene as insertion sites

If the ITR region, the VEGF gene, the PK gene, the gene coding for 10kDa protein or the intergenic region between the PK gene and the HD1R gene is to be used as an insertion site in a PPV, the corresponding Regions of the PPV genome, which include the insertion sites isolated. For this purpose, the corresponding regions of the PPV genome are cloned.

2.2.1 Cloning of the VEGF gene

1. a. Das VEGF-Gen wird beispielsweise über eine Polymerase-KettenReaktion (PCR) amplifiziert. Die für diese Reaktion notwendigen Start-Sequenzen (Primer) werden aus der im Sequenz Protokoll ID No: 1 dargestellten DNA-Sequenz des VEGF-Gens abgeleitet. Das erhaltene Amplifikat wird bevorzugt anschließend kloniert.
2. b. Bevorzugt wird die Region, die das VEGF-Gen und seine flankierenden Genomabschnitte enthält, über Fragmentierung des PPV-Genoms und Isolierung sowie Klonierung des oder der entsprechenden Genom-Fragmente gewonnen. Hierfür wird das gereinigte Genom des Virus wie in #2.1.2 beschrieben, bevorzugt mit dem Restriktionsenzym HindIII, geschnitten. Zur Identifizierung des oder der Genomfragmente, das oder die das VEGF-Gen und seine flankierenden Genomabschnitte tragen, werden bevorzugt die nach dem Enzymverdau erhaltenen Genom-Fragmente durch elektrophoretische oder chromatographische Verfahren aufgetrennt.
   Elektrophoretische Auftrennungen in Agarose bzw. Polyacrylamid werden nach Standardmethoden durchgeführt, die in
   • Current Protocols in Molecular Biology 1987-1988, Verlag Wiley-Interscience,1987
   • A Practical Guide to Molecular Cloning, Perbal, 2^nd edition, Verlag Wiley Interscience,1988
   • Molecular Cloning, loc. cit.
   • Virologische Arbeitsmethoden, Band III, Gustav Fischer Verlag, 1989
   beschrieben sind.
   Die Identifizierung der Genom-Fragmente, die das VEGF-Gen und seine flankierenden Sequenzen tragen, erfolgt beispielsweise durch Hybridisierung mit definierten Nukleinsäuresonden. Die aufgetrennten Genomfragmente werden hierzu auf Filter übertragen und mit VEGF-spezifischen und markierten Nukleinsäure-Sonden nach der Southern Blotmethode hybridisiert. Die Methode des Transfers der Genomfragmente und der Hybridisierung kann nach Standard-Protokollen, wie sie unter "Southern Blotting" in Molecular Cloning loc.cit. beschrieben sind, erfolgen. Die als Sonden einsetzbaren Oligonukleotide oder Nukleinsäurefragmente lassen sich aus Sequenzprotokoll Seq ID No: 1 ableiten. Beipielsweise wird das TaqI-Subfragment (366 bp), das mittels Seq ID No: 1 identifiziert werden kann als Hybridisierungssonde eingesetzt.
   Die Genomfragmente, die nachgewiesenermaßen Teile oder bevorzugt das gesamte VEGF-Gen und die flankierenden Genomabschnitte enthalten, werden isoliert und kloniert. Die elektrophoretische Isolierung des oder der entsprechenden Genomfragmente erfolgt beispielsweise über Elektroelution oder mittels "Low-Melting-Agarose"-Verfahren aus dem entsprechenden Gelbereich.

The gene encoding VEGF is located on the genome of the PPV and subsequently isolated in part or in its entirety with its flanking genome sections. For this purpose, the PPV is preferably propagated according to # 1 and the genome is purified according to # 2.1.1.

1. a. The VEGF gene is amplified, for example, via a polymerase chain reaction (PCR). The starting sequences (primers) necessary for this reaction are derived from the DNA sequence of the VEGF gene shown in the sequence protocol ID No: 1. The amplicon obtained is preferably subsequently cloned.
2. b. Preferably, the region containing the VEGF gene and its flanking genome segments is obtained via fragmentation of the PPV genome and isolation and cloning of the corresponding genome fragment (s). For this, the purified genome of the virus is cut as described in # 2.1.2, preferably with the restriction enzyme HindIII. To identify the genome fragment (s) carrying the VEGF gene and its flanking genome segments, preferably the genome fragments obtained after enzyme digestion are separated by electrophoretic or chromatographic techniques.
   Electrophoretic separations in agarose or polyacrylamide are carried out according to standard methods which are described in
   • Current Protocols in Molecular Biology 1987-1988, published by Wiley-Interscience, 1987
   • A Practical Guide to Molecular Cloning, Perbal, 2 ^nd edition, Wiley Interscience, 1988
   • Molecular Cloning, loc. cit.
   • Virological Working Methods, Volume III, Gustav Fischer Verlag, 1989
   are described.
   The identification of the genome fragments carrying the VEGF gene and its flanking sequences, for example, by hybridization with defined nucleic acid probes. For this purpose, the separated genomic fragments are transferred to filters and hybridized with VEGF-specific and labeled nucleic acid probes according to the Southern blot method. The method of transfer of genomic fragments and hybridization can be carried out according to standard protocols as described under "Southern blotting" in Molecular Cloning loc. are described done. The oligonucleotides or nucleic acid fragments which can be used as probes can be derived from Sequence Listing Seq ID No: 1. For example, the TaqI subfragment (366 bp), which can be identified by Seq ID No: 1, is used as the hybridization probe.
   The genomic fragments that have been shown to contain portions or preferably the entire VEGF gene and the flanking genome segments are isolated and cloned. The electrophoretic isolation of the corresponding genome fragments or, for example, by electroelution or by "low-melting agarose" method from the corresponding gel area.

For cloning the VEGF gene, the genomic fragments prepared above are inserted into bacterial or eukaryotic vectors. Particularly preferred are first bacterial plasmid or phage vectors. For insertion of the genomic fragment, double-stranded plasmid or phage vector DNA molecules are treated with restriction enzymes to form suitable ends for insertion.

Plasmids are known plasmids, e.g. pBR322 and its derivatives, e.g. pSPT18 / 19, pAT153, pACYC184, pUC18 / 19, pSP64 / 65.

As phage vectors are used e.g. the known variants of the phage lambda, e.g. -ZAP, -gt 10/11 or phage M13mp18 / 19.

The usable restriction enzymes are known e.g. from Gene Vol. 92 (1989) Elsevier Science Publishers BV Amsterdam.

The restriction enzyme treated plasmid or phage vector is incubated with an excess of the DNA fragment to be inserted, e.g. in the ratio 5: 1, mixed and treated by means of DNA ligases, ligated into the vector. The ligation mixture is introduced into and propagates the plasmids or phages in prokaryotic or eukaryotic cells, preferably in bacteria (e.g., Escherichia coli strain K-12 and its derivatives).

Transformation and selection of the bacteria is carried out as described in Molecular Cloning loc. sit. described.

The identity of the foreign DNA is preferably verified via hybridization experiments and more preferably via sequence analyzes. If necessary, subcloning is carried out.

2.2.2 Cloning of the protein kinase gene

The gene encoding the protein kinase is localized on the genome of the PPV and subsequently isolated in part or all with its flanking genome segments.

This area can be described as for the cloning of the VEGF gene. on the fragmentation of the PPV genome (cleavage sites see Fig. 1). preferably with subsequent cloning of the fragments, and selection of the fragments or clones which contain parts of the PK gene or preferably the entire PK gene with flanking DNA sequences of the PPV genome are obtained. DNA molecules used as primers of a PCR or as Probes can be used for a hybridization, can be derived from the sequence of Protocol ID No: 2 or ID No: 9.

If necessary, subcloning is carried out.

2.2.3 Cloning of the Gene Encoding the 10kDa Protein

The gene encoding the 10kDa protein is located on the genome of the PPV and partially or preferably entirely with its flanking PPV, according to the procedure detailed for the VEGF gene and the PK gene (above) Genome sequences isolated.

In this case, the region of the PPV genome containing the gene for the 10kDa protein or parts thereof is preferably obtained by PCR and / or cloning and identification and selection of the appropriate clones.

DNA molecules that can be used as primers of a PCR or as probes for hybridization can be found in reference # 8. If necessary, subcloning is carried out.

2.2.4 Cloning, the inverted-terminal repeat region or the genome section, which lies between the PK gene and the HD1R gene

The procedure corresponds to the detailed cloning of the VEGF gene (above). The DNA molecules that can be used to isolate the corresponding regions as primers of a PCR or as probes for hybridization are described in the Sequence Protocol ID No: 4 (ITR region) and No: 7 (region between PK gene and HDIR Gene).

3. Construction of Insertion Plasmids or Deletion Plasmids.

Based on the localized and cloned genomic fragments of PPV identified in accordance with Section 2, so-called insertion plasmids are produced, which can be used to insert foreign DNA into the genome of the PPV. The insertion plasmids carry the foreign DNA to be inserted into the PPV flanked by genome segments of the PPV. There are several ways to prepare the insertion plasmids: As examples may be mentioned here:

3.1 Identification or production of unique restriction sites in the cloned genomic fragments obtained according to 2.1.4 or 2.2 and insertion of foreign DNA

Only one-time, i. singular restriction sites, for example (see 2.1.3) can be identified the identified PPV-nucleotide sequences.

Synthesized oligonucleotides carrying new unique restriction enzyme cleavage sites can be incorporated into these.

The resulting plasmids are propagated and selected as described above.

Alternatively, by "PCR" as described by Jacobs et al. (Ref. # 12), new unique restriction enzyme recognition sites are incorporated into the PPV genome fragments.

The identified and / or produced unique restriction enzyme recognition sites serve to insert foreign DNA into the PPV genome.

The insertion of the foreign DNA is carried out by known methods (Ref. # 11).

3.2 Deletion of Genome Sequences in the Cloned Genome Fragments and Insertion of Foreign DNA

The deletion of subfragments from the cloned PPV genome fragments can be carried out, for example, by treatment with restriction enzymes which preferably have more than one, particularly preferably 2, recognition sites have. After the enzyme treatment, the fragments obtained are isolated, as described above, for example by electrophoresis, and the corresponding fragments are reassembled by ligase treatment. The resulting plasmids are propagated and the deleted plasmids are selected.

Alternatively, a unique restriction enzyme recognition site on the PPV genomic fragment is used as a starting point for bidirectional degradation of the fragments with an endonuclease, for example the enzyme Bal31. The size of the deletion can be determined by the duration of the action of the enzyme and checked by gel electrophoresis. Synthetic oligonucleotides, as described under 3.1 (above), are ligated to the newly formed fragment ends.

Foreign gene transfer into the PPV genome is only successful in a small percentage of the total PPV population.

Therefore, for the separation of recombinant from wild-type PPV selection systems are necessary (Ref # Moss).

Preferably, the gpt selection system is used, which is based on the guanyl phosphoribosyl transferase gene of E. coli. This gene, expressed in a eukaryotic cell, mediates resistance to mycophenolic acid, an inhibitor of purine metabolism. Its use in the construction of recombinant vector viruses has been described several times (see reference # 16 / # 17).

4. Construction of a recombinant PPV according to 1 to 12.

1. a. Gleichzeitige Transfektion der Insertions- oder Deletionsplasmid-DNA und Infektion mit PPV in geeigneten Wirtszellen,
2. b. Transfektion der Insertions- oder Deletionsplasmid- DNA und anschließende Infektion mit dem PPV in geeigneten Wirtszellen,
3. c. Infektion mit dem PPV und anschließende Transfektion mit der Insertions-oder Deletionsplasmid-DNA in geeigneten Wirtszellen.

The insertion of foreign DNA into the PPV genome is done by:

1. a. Simultaneous transfection of the insertion or deletion plasmid DNA and infection with PPV in appropriate host cells,
2. b. Transfection of the insertion or deletion plasmid DNA and subsequent infection with the PPV in suitable host cells,
3. c. Infection with the PPV and subsequent transfection with the insertion or deletion plasmid DNA in appropriate host cells.

1. 1. Infektion mit PPV:
   • Für die Herstellung der PPV mit Fremd-DNA werden Zellkulturen bevorzugt, die eine gute Virusvermehrung und eine effiziente Transfektion erlauben, beispielsweise die permanente Rindernierenzelle BK-K1-3A.
2. 2. Herstellung der Insertions- oder Deletionsplasmid-DNA:
   • Die nach den vorher beschriebenen Verfahren erhaltenen transformierten Zellen, beispielsweise Bakterien, die Insertions-oder Deletionsplasmide enthalten, werden vermehrt und die Plasmide in an sich bekannter Weise aus den Zellen isoliert und weiter gereinigt. Die Reinigung erfolgt z.B. durch eine isopyknische Zentrifugation im Dichtegradienten von z.B. CsCl. oder durch Affinitätsreinigung an kommerziell erhältlichen Silikapartikeln.
3. 3. Transfektion:
   • Zur Transfektion wird bevorzugt gereinigte Plasmid-DNA zirkular oder linearisiert eingesetzt. Die Reinigung erfolgt z.B. wie unter Abschnitt 2 (oben) angegeben.
4. 4. Kultivierung transfizierter und infizierter Zellen
   Die Zellen werden nach den oben beschriebenen Methoden kultiviert. Beim Auftreten eines zytopathogenen Effektes wird das Kulturmedium entfernt, gegebenenfalls durch Zentrifugation oder Filtration von Zelltrümmern befreit und gegebenenfalls gelagert sowie nach den herkömmlichen Methoden der Einzel-Plaque-Reinigung von Viren aufgearbeitet.

The methods of suitable methods are known. The transfection can be carried out by known methods such as calcium phosphate technique, liposome-mediated transfection or electroporation (see Ref. # 18).

1. 1. Infection with PPV:
   • For the production of PPV with foreign DNA, cell cultures are preferred which allow good virus replication and efficient transfection, for example the permanent bovine kidney cell BK-K1-3A.
2. 2. Preparation of Insertion or Deletion Plasmid DNA:
   • The transformed cells obtained by the previously described methods, for example bacteria which contain insertion or deletion plasmids, are propagated and the plasmids are isolated from the cells in a manner known per se and further purified. The purification is carried out, for example, by an isopycnic centrifugation in the density gradient of, for example, CsCl. or by affinity purification on commercially available silica particles.
3. 3. Transfection:
   • For transfection, purified plasmid DNA is preferably used in a circular or linearized manner. The cleaning takes place eg as described in section 2 (above).
4. 4. Cultivation of transfected and infected cells
   The cells are cultured according to the methods described above. When a cytopathogenic effect occurs, the culture medium is removed, optionally freed by centrifugation or filtration of cell debris and optionally stored and worked up by the conventional methods of single-plaque purification of viruses.

The following procedure is used in the production of recombinant PPV:

Confluent adult BK-KL-3A cells are infected with an infection dose of 0.001 to 5, preferably 0.1 MOI (multiplicity of infection). Two hours later, the infected cells are, for example, with DNA (2-10 ug) of the plasmid pMT-10 either by the CaPO ₄ glycerol shock method or using a transfection kit according to the manufacturer (DOSPER, Boehringer-Manaheim) transfected. Subsequently, these cell cultures are incubated with medium for 3 to 6 days at 37 ° C, 5% CO ₂ atmosphere until cpe or plaque formation is visible.

1. a. Nachweis der Fremd-DNA, z.B. durch DNA/DNA-Hybridisierungen
2. b. Die Amplifikation der Fremd-DNA mittels der PCR
3. c. Expression der Fremd-DNA mit Hilfe der rekombinanten Viren

The identification of recombinant PPV occurs depending on the inserted foreign DNA eg by:

1. a. Detection of foreign DNA, eg by DNA / DNA hybridizations
2. b. The amplification of the foreign DNA by PCR
3. c. Expression of foreign DNA using the recombinant viruses

to a.

For this purpose, the DNA is obtained from the virus in question and hybridized with nucleic acid which is identical at least in part to the inserted foreign DNA.

The individual plaque-purified and identified as recombinants PPV are preferably tested again for the presence and / or expression of the foreign DNA. Recombinant PPV stably containing and / or expressing the foreign DNA are available for further use.

to c.

The expression of the foreign DNA at the protein level can be detected by, for example, infection of cells with the virus and subsequent immunofluorescence analysis with specific antibodies to that of the Foreign DNA encodes protein or by immunoprecipitation or Western blotting with antibodies against the foreign DNA encoded protein from the lysates of infected cells.

Expression of foreign DNA at the RNA level can be accomplished by detection of the specific transcripts. For this purpose, RNA is isolated from virus-infected cells and hybridized with a DNA probe which is at least partially identical to the inserted foreign DNA.

Examples

The following examples describe a region of the PPV genome suitable for the insertion and expression of homologous and heterologous genes or parts thereof. Suitable genomic fragments are contained on HindIII fragment I of PPV ovis strain D1701 (and its respective derivatives) (Sequence Protocol ID No: 8 and ID No: 12).

1. Cloning of HindIII Fragment I:

The resulting DNA fragments were separated by agarose gel electrophoresis after cleavage of purified viral DNA with the Hind III restriction enzyme, the excised fragment of about 5.6 kbp and I by means of the Qiaex ^® method (Qiagen) and purified. This DNA fragment was cloned by standard techniques (Maniatis et al.) Into HindIII digested and CIP (calf intestinal phosphatase) treated vector plasmid pSPT18 (Boehringer, Mannheim). The resulting recombinant plasmids pORF-1 and pORF-2 differ only in the orientation of the insert. The preparation of a restriction map allowed further subcloning, as shown in FIG. All recombinant plasmid DNAs were tested by Southern blot hybridization for restriction digested viral or plasmid DNA to verify their identity and viral origin.

Second DNA sequencing

DNA sequencing was accomplished by using double stranded DNA of the various recombinant plasmids and SP6 and T7 specific primers that bind to both ends of the cloning site of the vector plasmid pSPT18. The Sanger dideoxy chain termination method was performed according to the manufacturer's recommendations (Pharmacia Biotech) in the presence of ³⁵ S- [α] -dATP and T7 DNA polymerase. According to the obtained DNA sequence, a plurality of oligonucleotides were synthesized and used for sequencing both strands of HindIII fragment I. To resolve sequencing artifacts or band compression due to the relatively high content of the viral DNA insert (64.78%), 7-deaza-GTP was used and the sequencing products separated, if necessary, in formamide containing denaturing polyacrylamide gels.

Third Identification of potential genes

• 3.1 Ein ORF mit Aminosäurehomologie (36,1 bis 38,3 % Identität; 52,8 bis 58,6 % Ähnlichkeit) zum vaskulären endothelialen Wachstumsfaktor (VEGF) verschiedener Säugerspezies (z.B. Maus, Ratte, Meerschweinchen, Rind, Mensch) als auch zu dem VEGF-Gen-Homolog, der vor kurzem in den PPV-Stämmen NZ-2 und NZ-7 beschrieben wurde (Lit. #6), wurde gefunden. Ein entsprechendes Genhomolog ist bei anderen Pockenviren, wie verschiedenen Orthopoxviren, nicht bekannt. Dieser ORF, der als VEGF bezeichnet wird, umfaßt 399 Nucleotide und kodiert ein Polypeptid mit 132 Aminosäuren mit einem berechneten Molekulargewicht von 14,77 kDa. Transkriptionsanalysen unter Verwendung von Northern-Blothybridisierung, RNA-Schutzversuchen und Primerextensionstests mit gesamter oder Oligo (dT) - selektierter RNA, die aus infizierten Zellen isoliert wurde, belegten die Expression von VEGF als frühes Gen ab etwa 2 Stunden nach der Infektion (p.i.). Es wurde gefunden, daß die spezifische mRNA 422 bis 425 Basen abdeckt, beginnend direkt stromabwärts einer Sequenz, die 100 % Homologie mit dem kritischen Bereich eines Consensusmotivs zeigt, das typisch für Promotoren früher Gene des Vaccinia-Virus ist. Das 3'-Ende der VEGF-mRNA konnte in eine Consensussequenz kartiert werden, die frühen Transkripten von z.B. Vaccinia-Virus-Genen gemeinsam ist. Durch Northern-Blothybridisierung wurde die Größe dieser mRNA auf ungefähr 500 Basen geschätzt, was auf einen Poly(A)-Abschnitt mit einer Länge von etwa 100 Basen hinweist.
• 3.2 Ein weiterer ORF wurde gefunden, der für eine potentielle Proteinkinase (PK) mit Homologie zu einem entsprechenden Gen kodiert, das in mehreren Orthopoxviren (z.B. Vaccinia-, Variola- oder Shope-Fibroma-Virus) vorkommt und als F10L bekannt ist. Dieses Gen-Homolog wird im Infektionszyklus von PPV-Stamm D1701 spät transkribiert (ab 12 bis 16 Stunden p.i.). Der Transkriptionsstartpunkt befindet sich kurz stromabwärts eines Bereichs, der eine hohe Homologie zu bekannten Promotoren später Gene des Vaccinia-Virus zeigt.
• 3.3 Weitere mögliche ORFs wurden gefunden, die bisher keine auffälligen Homologien zu bekannten Gensequenzen zeigen. Von besonderem Interesse ist ein potentielles Gen, das als Gen HD1R bezeichnet wird (Abb. 1). Analysen wie die oben beschriebenen zeigten die Transkription einer spezifischen frühen mRNA mit einer Größe von ca. 1,6 kb.
• 3.4 Schließlich wurde mittels Computer ein ORF gefunden, der das 3'-Ende von F10L und das 5'-Ende von VEGF überlappt (F9L, Abb. 1). Der Sequenzvergleich zeigte Homologie mit dem F9L-Gen des Vaccinia-Virus.
• 3.5 Verglichen zu bekannten DNA Sequenzen der Orf Stämme NZ-2 und NZ-7 ließ sich der Beginn der sogenannten ITR. Region im Genom von D 1701 bei Nukleotidposition 1611 des HindIII-Fragments I festlegen. Bei der ITR-Region handelt es sich um einen am Ende des Pockenvirusgenoms auftretenden Sequenzbereich, der am anderen Genomende in umgekehrter Orientierung ebenfalls vorliegt und daher als "inverted repeat region" (ITR) bezeichnet wird (siehe Sequenzprotokoll ID No: 4). Der Befund des Sequenzvergleichs deckt sich mit Versuchen zur Genomlokalisation des in pORF-1 klonierten Hindin-Fragments I von D1701. Die Kartierung dieses Fragments und des vermutlich identischen HindIII-Fragments H ist in Abb. 2 dargestellt. Hieraus läßt sich schließen, daß die ITR des D1701 Genoms ca. 2,6 kbp umfaßt.
  Versuche zur Bestimmung des 3' Endes der VEGF mRNA von D1701 enthüllten, daß zwischen ca. 40 und 220 bp nach dem Übergang zur ITR in der ITR mindestens eine weitere virusspezifische RNA startet. Das entsprechende Gen wurde aufgrund der Aminosäurehomologie zu NZ-2 als ORF3 bezeichnet (Abb. 1). Vor dem putativen 5' Ende der ORF3-mRNA befindet sich eine Konsensussequenz, die typisch für einen frühen Promotor von Pockenviren ist. Homologie zu anderen bekannten Genen konnte bisher nicht gefunden werden.

A computer-assisted analysis of the DNA sequence obtained (Sequence Listing ID No: 8 and ID No: 12) revealed several possible open reading frames (ORF). The amino acid sequence deduced from these ORFs was used for gene homology searches (eg program GCG). As a result, significant amino acid homologies were detected with the following genes (see also Fig. 1).

• 3.1 An ORF with amino acid homology (36.1 to 38.3% identity, 52.8 to 58.6% similarity) to the vascular endothelial growth factor (VEGF) of various mammalian species (eg mouse, rat, guinea pig, bovine, human) as well the VEGF gene homologue recently described in PPV strains NZ-2 and NZ-7 (ref. # 6) was found. A corresponding gene homologue is unknown in other smallpox viruses, such as various orthopoxviruses. This ORF, designated VEGF, comprises 399 nucleotides and encodes a 132 amino acid polypeptide with a calculated molecular weight of 14.77 kDa. Transcriptional analyzes using Northern blot hybridization, RNA protection experiments and primer extension testing with whole or oligo (dT) -selected RNA isolated from infected cells demonstrated expression of VEGF as an early gene from about 2 hours after infection (pi). The specific mRNA was found to cover 422 to 425 bases, starting just downstream of a sequence showing 100% homology with the critical region of a consensus motif typical of promoters of early vaccinia virus genes. The 3 'end of the VEGF mRNA could be mapped into a consensus sequence common to early transcripts of vaccinia virus genes, for example. Northern blot hybridization estimated the size of this mRNA to be approximately 500 bases, indicating a poly (A) segment about 100 bases in length.
• 3.2 Another ORF was found encoding a potential protein kinase (PK) with homology to a corresponding gene found in several orthopoxviruses (eg vaccinia, variola or shope fibroma virus), known as F10L. This gene homologue is late transcribed in the infection cycle of PPV strain D1701 (from 12 to 16 Hours pi). The transcription start site is located just downstream of a region that shows high homology to known promoters of late vaccinia virus genes.
• 3.3 Further possible ORFs have been found which so far show no conspicuous homologies to known gene sequences. Of particular interest is a potential gene called the HD1R gene (Figure 1). Analyzes such as those described above demonstrated the transcription of a specific early mRNA of about 1.6 kb in size.
• 3.4 Finally, an ORF was found by computer overlapping the 3 'end of F10L and the 5' end of VEGF (F9L, Fig. 1). Sequence comparison showed homology with the vaccinia virus F9L gene.
• 3.5 Compared to known DNA sequences of the Orf strains NZ-2 and NZ-7, the so-called ITR began. Region in the genome of D 1701 at nucleotide position 1611 of HindIII fragment I. The ITR region is a sequence region occurring at the end of the poxvirus genome which is also present in reverse orientation at the other end of the genome and is therefore designated as "inverted repeat region" (ITR) (see Sequence Listing ID No: 4). The finding of the sequence comparison coincides with experiments on the genome localization of the pORF-1-cloned Hindin fragment I of D1701. The mapping of this fragment and the probably identical HindIII fragment H is shown in FIG. It can be concluded that the ITR of the D1701 genome comprises about 2.6 kbp.
  Experiments to determine the 3 'end of D1701 VEGF mRNA revealed that at least one additional virus-specific RNA starts in the ITR between about 40 and 220 bp after the ITR transition. The corresponding gene was designated ORF3 because of the amino acid homology to NZ-2 (Figure 1). Prior to the putative 5 'end of ORF3 mRNA, there is a consensus sequence that is typical of an early promoter of poxviruses. Homology to other known genes could not be found so far.

4th Introduction of DNA sequences in the HindIII fragment I

The possibility of using the HindIII DNA fragment described (cloned in the plasmids pORF-1 and pORF-2) for the introduction of homologous or heterologous DNA sequences was developed. In the following, three different strategies were used to achieve this goal.

For the following examples, the plasmid pGSRZ was constructed which contains the functional LacZ gene of E. coli under the control of the vaccinia virus 11K promoter. For this purpose, the relevant DNA parts of the plasmid pUCIILZ (see reference # 7) were isolated and cloned into the plasmid pSPT18. This functional 11K-LacZ gene combination (hereinafter referred to as LacZ cassette) can be obtained by isolating a 3.2 kbp SmaI-SalI fragment from pGSRZ (Figure 3).

Construction of the selection cassettes

Starting from the plasmids pGSRZ (containing the lacZ gene under the control of the vaccinia virus promoter P _11K ), pMT-1 (containing the PPV VEGF promoter) and pMT5 (containing the E. coli gpt gene), various so-called selection cassettes were constructed, such as shown in Figure No: 7. Synthetic complementary oligonucleotides representing the sequence of the VEGF promoter (P _VEGF ) were prepared and inserted into the SmaI site of pSPT18 (pMT-1). Subsequently, plasmids pMT-2 and pMT-4 were prepared by removing the LacZ gene from p18Z (obtained by insertion of the BamHI fragment from pGSRZ into pSPT18) or the gpt gene from pMT-5 by BamHI digestion, and into pMT-1 was inserted (Figure 7).

The functional gpt gene was amplified by PCR from the gpt plasmid pMSG (Pharmacia Biotech) and cloned into the vector pCRII by so-called TA cloning according to the instructions of the manufacturer (Invitrogen Inc.).

In the following, as shown schematically in Fig. 7, double selection cassettes could be constructed which in the combinations and orientations shown express the LacZ gene or gpt gene under the control of the 11K promoter and / or the P _VEGF .

According to Example XX (LacZ-VEGF deletion) or YY (intergenic Ba131), these selection cassettes can be inserted into the various PPV orf DNA plasmids after appropriate restriction enzyme cleavage and isolation. After insertion of a double selection cassette into the described plasmid pdV-500, which has a 312 bp deletion of the VEGF gene of PPV D 1701, the plasmid pMT-10 was constructed. By this construction, the functional LacZ and gpt genes were substituted for the deleted VEGF ORF. After transient expression assays, the activity of the LacZ gene was demonstrated in PPV D1701-infected cells (not shown) so that further selection was made for gpt- and lacZ-expressing VEGF deletion mutants of the D1701.

4.1 Insertion into intergenic, non-coding regions

Identification or creation of new unique restriction sites located in an intergenic noncoding part. These sites are then used to insert foreign DNA sequences encoding functional and detectable gene products (e.g., the LacZ gene, from E. coli) or portions thereof.

Example 4.1.1

The plasmid pORF-PB (Figure 1) contains a single NruI site located between the protein kinase (F10L) and the HD1R gene. After linearization of pORF-PB with this restriction enzyme, the LacZ cassette (after fill-in reaction) was blunt-end ligated. Recombinant plasmids containing the functional LacZ gene were cleaved, e.g. was selected with BglI and the correct insertion demonstrated by Southern blot hybridization with a LacZ-specific probe as well as by partial DNA sequencing of the LacZ and PPV DNA transition.

Example 4.1.2

The same procedure as described in the above example was used downstream of the VEGF gene (cleavage at the BstEII site, Fig. 1) and in the potential gene ORF 3 in the ITR region (partial degradation with XbaI, to prevent cleavage of the XbaI site in the pSPT18 cloning sites).

Example 4.1.3

To introduce a novel, unique restriction site into any desired location of cloned PPV DNA fragments, the technique of PCR mutagenesis can be used (see Fig. 10, ref. # 12). For this purpose, two PCR reactions are carried out separately using the primer pair E1 + EV2 (PCR A) and EV1 + XB (PCR B), respectively. All primers cover 25 nucleotides that are identical to the PPV DNA sequence at the indicated sequence positions. While primer XB contains the authentic sequence, e.g. representing the XbaI site (Figure 1), a new EcoRI site was inserted into primer E1 at the 5 'end and a new EcoRV site was inserted into primers EV1 and EV2 (which are complementary to each other). The EcoRV site, which is absent in the entire sequence of pORF-1 or -2, was placed at the site chosen for the introduction of the LacZ cassette. The PCR products obtained from reactions A and B are purified, denatured to single strands and mixed together under reassociation conditions; then they are used for the last PCR C reaction. As primers E1 and XB are now used to extend in this example the left 793 bp of pORF-1. After gel isolation and purification, the resulting PCR product from reaction C is cleaved with EcoRI and XbaI and then ligated to the EcoRI and XbaI digested plasmid pORF-XB. As a result, the plasmid pORF-1EV (Figure 5) contains the EcoRV restriction site at the desired position; it can then be used for linearization and ligation with the LacZ cassette.

In addition, mismatch primers containing defined base changes or a single base deletion may be used for the method described in this example to generate, for example, translation stop codons or amino acid deletions at any desired location in the viral DNA sequence.

4.2 Intragenic insertion without deletion of Orf sequences

New or additional sequences can be introduced into the coding sequences of one of the described ORFs after cleavage to restriction sites which occur only once in the selected gene.

Example 4.2.1

The unique XcmI site, located in the right-hand part of the gene homolog F10L, was used to linearize the plasmid pORF-1. Both the LacZ cassette and the cleaved pORF-1 DNA were blunt-ended by T4 or Klenow DNA polymerase, and competent E. coli bacteria (DHSαF ') were used for transformation. The resulting bacterial colonies were tested for positive recombinant plasmids by colony filter hybridization using a LacZ-specific probe, and then the respective plasmid DNAs were subjected to restriction digestion.

Example 4.2.2

The VEGF coding region contains a single StyI site used as described above to insert the LacZ cassette.

4.3 Deletion of viral sequences

The following examples describe the removal of both coding (intragenic deletions) and non-coding (intergenic deletions) regions:

Example 4.3.1

By using restriction enzymes, defined portions of HindIII DNA fragment I are deleted to replace the deleted viral sequences by inserting foreign genes or portions thereof. This was done by cleavage of the plasmid pORF-PA with the restriction enzyme NruI (at a position in the ITR region, Fig. 1), wherein a 396 bp fragment was deleted. After a padding reaction, the LacZ cassette was ligated as described above. Illustration Figure 4 shows schematically the deletion of the 396 bp fragment from pORF-PA and the insertion of the LacZ cassette. Figures 5 and 6 show the thus constructed deletion / insertion plasmids pCE4 and pCE9 derived from pORF-PA.

Example 4.3.2

Single restriction sites in the HindIII DNA fragment were used as starting points to effect bidirectional deletion of sequences by the action of endonuclease Bal31, as schematically outlined in Figure 6. For restriction digestion, the plasmid pORF-PA used the enzyme StyI and the plasmid pORF-1 or pORF-XB the enzyme XcmI to open the genes which code for VEGF or protein kinase F10L (FIGS. 1 and 6). After addition of exonuclease Ba131, equal parts of the reaction were taken every 2 minutes and the reaction was stopped. Cleavage of the time values e.g. with the restriction enzyme BglI and subsequent gel electrophoresis allowed to estimate the size of the DNA segment deleted by Bal31. Mixtures from the appropriate times were then used to close the DNA ends by a make-up reaction. Two complementary oligonucleotides representing novel SmaI, SalI and EcoRV unique restriction sites were hybridized and the resulting double-stranded primer molecules (termed "EcoRV" linkers) ligated to the blunt-ended Ba131 products. After transformation of bacteria, plasmid DNA was isolated and cleaved with EcoRV containing no recognition site in the DNA sequence of the PPV HindIII fragment I. Therefore, each plasmid DNA with an Eco RV site contained the inserted linker sequence and was then used for ligation of the blunt-ended LacZ cassette into the new Eco RV site. The exact size of the generated DNA deletion in each resulting recombinant plasmid DNA could be determined by sequencing with the single-stranded EcoRV linkers as well as with suitable LacZ gene-specific primers.

5th Detection and identification of the VEGF gene in other parapoxviruses

Knowledge of the DNA region encoding the VEGF in D1701 allows the production of specific DNA probes and PCR primers to identify this gene in other parapoxvirus strains, which may have extremely different restriction profiles. For this purpose were the following options tested: (i) isolation of a TaqI subfragment (366 bp in size) of pORF-PA as a hybridization probe, which is the midportion of the D1701 VEGF gene; (ii) amplification of the complete VEGF ORF using appropriate synthetic primers followed by plasmid cloning of the PCR product; (iii) Use of primers covering various parts of the VEGF gene were used for PCRs in the presence of PPV DNAs as templates as well as specific hybridization probes.

Following radioactive labeling, these probes were successfully used for Southern and Dot / Spot blot hybridization with the genomic DNA of various isolates and strains of Parapox ovis, Parapox bovis 1 (Bovine papular stomatitis, BPS) and Parapox bovis 2 (Melker nodules). Southern blot hybridization revealed VEGF-positive signals with defined PPV-DNA fragments, allowing further detailed mapping of the VEGF gene of the various PPV.

In addition, the same probes can be used for comparative RNA analyzes, such as Northern blot hybridization, to test the expression of potential VEGF genes in other PPV strains.

6th Generation of D1701 recombinants

1. (a) Das Zell-Lysat gewonnen, eine Verdünnungsreihe hergestellt und ein Plaque-Test auf BK-KL-3A Zellen durchgeführt. Das zugegebene Agarose-Mediumgemisch enthielt 0,3 mg/ml Bluo-Gal (Life Science GIBCO-BRL), um blaue Plaques zu identifizieren, die LacZ-exprimierende, MPAresistente D1701-Rekombinanten enthielten.
2. (b) Nach Bildung von Einzelplaques wurde das in (a) beschriebene Agarose-Bluo Gal-Gemisch zugegeben und blau gefärbte Einzelplaques gepickt.

Confluent adult BK-KL-3A cells were infected with an infection dose of 0.1 moi (multiplicity of infection). Two hours later, the infected cells were transfected, for example, with DNA (2 to 10 μg) of the plasmid pMT-10 either by the CaPO ₄ glycerol shock method or using a transfection kit according to the manufacturer (DOSPER, Boehringer-Mannheim). Subsequently, these cell cultures were incubated with selective medium (HAT medium + MPA mycophenolic acid - xanthine - 5% FCS) for 3 to 6 days at 37 ° C, 5% CO ₂ atmosphere until cpe or plaque formation became apparent. Depending on the severity of the virus-related cpe:

1. (a) Obtain the cell lysate, prepare a dilution series, and perform a plaque assay on BK-KL-3A cells. The added agarose medium mixture contained 0.3 mg / ml Bluo-Gal (Life Science GIBCO-BRL) to identify blue plaques containing LacZ-expressing, MPA-resistant D1701 recombinants.
2. (b) After formation of single plaques, the agarose-Bluo Gal mixture described in (a) was added and blue colored single plaques were picked.

The viruses obtained in (a) or (b) are used as described above for the infection of BK-KL-3A and subjected to at least two further plaque titrations and purifications until a 100% homogeneous recombinant virus population is present.

7th VEGF promoter

As outlined in Chapter 3.1, the D1701 VEGF gene is an early gene, the specific mRNA is transcribed in large quantities in D1701 virus-infected cells from two to four hours after infection at later times of infection. Therefore, the identified promoter region of the D1701 VEGF should be very useful for directing the expression of foreign genes or portions thereof in recombinant PPV viruses. The sequence comprising the VEGF promoter (35-40 nucleotides; Sequence Protocol ID No: 6) can be isolated by (i) PCR using the appropriate primers flanking the promoter region, (ii) subcloning the corresponding DNA sequence. Fragments or (iii) synthesizing the promoter sequence as an oligonucleotide. After linking the VEGF promoter to any gene of interest or to a DNA sequence, the resulting gene cassette can be used to produce recombinant PPV by any method as described in the preceding chapter.

8 . 10 kDa gene

Based on the published DNA sequence of the 10 kDa gene of the PPV strain NZ-2 (ref. # 8), a specific PCR for the detection of the 10 kDa gene of PPV was established. After carrying out the PCR with the synthetically prepared primers 10K-up (5-CAATATGGATGAAAATGACGG-3) and 10k-low (5-CAGACGGCAACACAGCG-3), amplification of a specific 297 base pair product was achieved. Subsequent cloning (TA cloning kit, Invitrogen Inc.) yielded plasmid pJS-1, which contained the 297 bp PCR product as EcoRI fragment. DNA sequencing of both DNA strands of the insert of pJS-1 demonstrated the presence of the 10kDa-specific sequence. Thereafter, D1701 encodes a 91 amino acid 10kDa protein that belongs to the of the PPV strain NZ-2 93.3% identical or 96.7% similar amino acids.

Localization of the 10kDa gene in the EcoRI fragment E (4.25 kbp) of the D1701 genome was by Southern blot hybridization with radiolabelled pJS-1. Thus, similar to NZ-2, this gene is located in the right part of the virus genome and includes the junction of HindIII fragments K and G (Figure 2).

The plasmids pDE-E1 and pRZ-E1 which contain the 4.25 kbp EcoRI fragment E of D1701 are used for the production of insertion and deletion plasmids in the 10 kDa gene (in principle as described above). The HindIII site (fragments KG) in the N-terminal portion of the 10kDa gene of D1701 (# 124- # 129 Sequence ID No: 11) can be used both for direct insertion of foreign DNA and for deletion (see Ba131 bidirectional digestion). of the 10 kDa gene. For the latter construction, the second HindIII site (the multiple cloning site of the vector plasmid pSPT18) was removed in plasmid pDE-E1. For this purpose, pSPT18 DNA was digested with HindIII, the HindIII site disrupted by Klenow treatment and religated. The 4.25 kbp EcoRI fragment E of D1701 was then cloned into the EcoRI site of this new vector plasmid pSPT18dH. The resulting plasmid pRZ-E1 (Figure 8) now has a unique HindIII site in the 10 kDa gene, which allows further simple manipulation.

Southern blot hybridization with pDE-E1 and pJS-1 as radiolabeled probes, as well as PCR assays, show that genomes of different PPV bovis 1 do not contain 10 kDa specific sequences. This indicates that the 10 kDa gene of PPV is not essential (Büttner M. et al., 1996, Ref # 10).

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   Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc. Natl. Acad. Sci. USA 89, 10847-10851.
8. 8. Naase, M., Nicholson, BH, Fraser, KM, Mercer, AA and Robinson, AJ 1991.
   An orf virus sequence showing homology to the 14K fusion protein of vaccinia virus. J. Gen.. Virol. 72, 1177-1181.
9. 9. Graham, FL and van der Eb, AJ 1973.
   A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467.
10. 10. Büttner, M., McInnes, C., von Einem, C., Rziha, HJ and Haig, D. 1996.
    Molecular discrimination of parapoxviruses from different species. 11th Poxvirus and Iridovirus meeting, 4-9. May, Toledo, Spain.
11. 11. Sambrock, J., Frisch, EF and Maniatis, T. 1989.
    Molecular Cloning, A laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Press.
12. 12. Jacobs, L., Rziha, H.-J., Kimman, TG, Gielkens, ALJ and von Oirschot, JT 1993.
    Deleting valine-125 and cysteine-126 in glycoprotein gl of pseudorabies virus strain NIA-3 decreases plaque size and reduces virulence for mice. Arch. Virol. 131, 251-264.
13. 13. Virological working methods, 1989.
    Biochemical and Biophysical Methods, VEB Fischer Verlag.
14. 14. Sharp, PA, Berk. AJ and Berger, SM 1980.
    Transcription maps of adenovirus. Meth. Enzymol. 65, 750-768.
15. 15. Watson, JD, Hopkins, NH, Roberts, JW, Seitz, JA and Weiner, AM 1987.
    Molecular Biology of the Genes, Benjamin / Cummins Publishing Company, Menlo Park.
16. 16. Falconer, FG and Moss, B. 1988.
    Escherichia coli gpt gene provides dominant selection for vaccine virus open reading frame expression vectors. J. Virol. 62, 1849-1854.
17. 17. Boyle, DB and Coupar, BE 1988.
    Construction of recombinant fowlpox viruses as vectors for poultry vaccines. Virus Res. 10, 343-356.
18. 18. Methods in Virology, Vol. VI, 1977.
    Edts. Maramorosch, K. and Koprowski H. Academic Press. New York, San Francisco.
19. 19. Hattori, M. and Sakaki Y. 1986.
    Dideoxy sequencing method using denaturated plasmid templates. Anal. Biochem. 152, 232-238.
20. 20. Chomczynski, P. and Sacchi, N. 1987.
    Single step method of RNA isolation by acid guanidinium-thiocyanate phenol-chloroform extraction. Ana. Biochem. 162, 156-159.

ID No: 1

Sequence ID No: 1 of the application shows the VEGF gene located on HindIII fragment I of strain PPV D1701.

Additional Information:

Earyl promoter: Nucleotides 50 to 64 mRNA start: Nucleotides 78 and 80, respectively mRNA Stop: Nucleotides 498 to 500 Translation start: Nucleotides 92 to 94 Translation stop: Nucleotides 488 to 490

ID No: 2

Sequence ID No: 2 of the application shows the protein kinase gene F10L (version 1) located on HindIII fragment I of strain PPV D1701

Additional Information:

Late promoter: Nucleotides 48 to 66 mRNA start: Nucleotides 74 to 78 Translation start: Nucleotides 94 to 96 Translation stop: Nucleotides 1738 to 1740

ID No: 3

Sequence ID No: 3 of the application shows the HD1R segment located on Hindin fragment I of strain PPV D1701.

ID No: 4

Sequence ID No: 4 of the application shows the ITR region located on HindIII fragment I of strain PPV D1701 and the ORF3 gene in this region.

Additional Information:

Start ITR region: Nucleotide 7 Early promoter: Nucleotides 18 to 33 ORF3 mRNA start: Nucleotides 40 to 41 ORF3 mRNA stop: Nucleotides 673 to 679 ORF3 translation start: Nucleotides 111 to 113 ORF3 translation stop: Nucleotides 562 to 564

ID No: 5

Sequence ID No: 5 of the application shows the F9L gene homolog located on HindIII fragment I of strain PPV D1701 (version 1).

Additional Information:

start codon: Nucleotides 48 to 50 stop codon: Nucleotides 861 to 863

ID No: 6

Sequence ID No: 6 of the application shows the VEGF promoter region located on HindIII fragment I of strain PPV D1701.

ID No: 7

Sequence ID No: 7 of the application shows the intergenic region located on HindIII fragment I of strain PPV D1701, located between the HD1R and the PKF10L gene. Putative translation stop HD1R: Nucleotides 25 to 27 Translation start PKF10L: Nucleotides 223 to 225

ID No: 8

Sequence ID No: 8 of the application shows the complete nucleotide sequence of HindIII fragment I (version 1) of PPV strain D1701.

ID No: 9

Sequence ID No: 9 of the application shows version 2 of the protein kinase F10L gene located on HindIII fragment I of strain PPV D1701.

Additional Information:

Late promoter: Nucleotides 48 to 66 RNA start signal: Nucleotides 72 to 80 mRNA start: Nucleotides 74 to 78 Translation start: Nucleotides 94 to 96 Translation stop: Nucleotides 1585-1588

ID No: 10

Sequence ID No: 10 of the application shows version 2 of the F9L gene homolog located on HindIII fragment I of strain PPV D1701.

Additional Information:

Translation start: Nucleotides 50 to 52 Translation stop: Nucleotides 722 to 724

ID No: 11

Sequence ID No: 11 of the application shows the 10 kDa gene located on EcoRI fragment E of strain PPV D1701.

Additional Information:

Translation start: Nucleotides 5 to 7 Translation stop: Nucleotides 275 to 277

ID No: 12

Sequence ID No: 12 of the application shows the complete nucleotide sequence of version 2 of HindIII fragment I of PPV strain D1741.

ID No: 13

Sequence ID No: 13 of the application shows version 3 of the protein kinase FIOL gene located on HindIII fragment I of strain PPV D1701.

Additional Information:

Late promoter: Nucleotides 48 to 66 RNA start signal: Nucleotides 72 to 80 mRNA start: Nucleotides 74 to 78 Translation start: Nucleotides 94 to 96 Translation stop: Nucleotides 1585-1588

ID No: 14

Sequence ID No: 14 shows the amino acid sequence of the PPV D1701 protein kinase F10L homolog (derived from Sequence ID No: 13).

ID No: 15

Sequence ID No: 15 shows the amino acid sequence of the PPV D1701 VEGF homolog (derived from Sequence ID No: 1).

ID No: 16

Sequence ID No: 16 shows the amino acid sequence of the PPV D1701 F9L homolog (derived from Sequence ID No: 10).

SEQUENCE LISTING

• (1) GENERAL INFORMATION:
  • (i) APPLICANT:
    • (A) NAME: BAYER AG
    • (B) STREET: Bayerwerk
    • (C) CITY: Leverkusen
    • (E) COUNTRY: Germany
    • (F) POSTAL CODE (ZIP): D-51368
    • (G) TELEPHONE: 0214/3061285
    • (H) TELEFAX: 0214/30 3482
  • (ii) TITLE OF INVENTION: Parapox viruses containing foreign DNA, their production and their use in vaccines
  • (iii) NUMBER OF SEQUENCES: 16
  • (iv) COMPUTER READABLE FORM:
    1. (A) MEDIUM TYPE: Floppy disk
    2. (B) COMPUTER: IBM PC compatible
    3. (C) OPERATING SYSTEM: PC-DOS / MS-DOS
    4. (D) SOFTWARE: Patent In Release # 1.0, Version # 1.30 (EPO)
• (2) INFORMATION FOR SEQ ID NO: 1:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 540 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 VEGF gene
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
    
• (2) INFORMATION FOR SEQ ID NO: 2:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 1740 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 protein kinase gene (version 1)
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
    
    
• (2) INFORMATION FOR SEQ ID NO: 3:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 1080 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701-HD1R gene region
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
    
    
• (2) INFORMATION FOR SEQ ID NO: 4:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 1616 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 ITR and ORF3 gene
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
    
    
• (2) INFORMATION FOR SEQ ID NO: 5:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 900 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 F9L Gene (Version 1)
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
    
• (2) INFORMATION FOR SEQ ID NO: 6:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 94 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 VEGF promoter
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
    
• (2) INFORMATION FOR SEQ ID NO: 7:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 250 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 intergene. Region between HD1R and protein kinase gene
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
    
• (2) INFORMATION FOR SEQ ID NO: 8:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 5515 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701- HIND III fragment I (version 1)
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
    
    
    
    
• (2) INFORMATION FOR SEQ ID NO: 9:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 1620 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 protein kinase gene F10L (version 2)
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
    
    
• (2) INFORMATION FOR SEQ ID NO: 10:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 780 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701-F9L gene, version 2
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
    
• (2) INFORMATION FOR SEQ ID NO: 11:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 297 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 10kD gene
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
    
• (2) INFORMATION FOR SEQ ID NO: 12:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 5519 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701, HindIII fragment I, version 2
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
    
    
    
    
    
• (2) INFORMATION FOR SEQ ID NO: 13:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 1742 base pairs
    2. (B) TYPE: nucleic acid
    3. (C) BEACHEDNESS: double
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D 1701 protein kinase gene F10L (version 3)
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
    
    
• (2) INFORMATION FOR SEQ ID NO: 14:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 497 amino acids
    2. (B) TYPE: amino acid
    3. (C) BEACHEDNESS: single
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: protein
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 protein kinase F10L
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
    
    
• (2) INFORMATION FOR SEQ ID NO: 15:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 132 amino acids
    2. (B) TYPE: amino acid
    3. (C) BEACHEDNESS: single
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: protein
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 VEGF protein
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
    
• (2) INFORMATION FOR SEQ ID NO: 16:
  • (i) SEQUENCE CHARACTERISTICS:
    1. (A) LENGTH: 224 amino acids
    2. (B) TYPE: amino acid
    3. (C) BEACHEDNESS: single
    4. (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: protein
  • (iii) HYPOTHETICAL: NO
  • (iv) ANTI-SENSE: NO
  • (vi) ORIGINAL SOURCE:
    1. (A) ORGANISM: Parapox ovis
    2. (B) STRAIN: D1701 protein F9L
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
    

List of pictures

Fig. 1

shows the physical map of HindIII fragment I from Orf D1701 in plasmids pORF-1 / -2. The thin arrows mark the identified mRNAs, the thick arrows ORFs.

Fig. 2

Figure 4 shows the physical map of Hind III recognition sites on the D1701 genome and the genes identified on Hind III fragment I, as well as a portion of the inverted terminal repeat region.

Fig. 3

shows plasmid pCE4. After NruI digestion, a 396bp fragment was replaced with the LacZ cassette.

Fig. 4

shows plasmid pCE9 into which, after linearization by XcmI cleavage, the LacZ cassette was inserted.

Fig. 5

schematically shows, as described in the text, the strategy for generating new singular interfaces by PCR.

Fig. 6

schematically shows the bidirectional truncation by the nuclease Bal31 with subsequent insertion of the EcoRV linker and LacZ cassette.

Fig. 7

shows the strategy for making the LacZ / gpt selection cassette:

• P _11K : Vaccinia 11k promoter
• P _VEGF : PPV VEGF promoter
• Sm: Sma I.
• B: Bam H1

Fig. 8

Figure 3 shows schematically the cloning strategy of EcoR1 fragment E of PPV D1701 containing the 10 kDa gene (as described in the text).

Claims (4)

1. Recombinantly prepared PPVs with insertions and/or deletions, containing a foreign antigen which can induce a pathogen-specific protection.
2. Recombinantly prepared PPVs with insertions and/or deletions, containing a cytokine.
3. Recombinantly prepared PPVs according to Claim 1 or 2, containing insertions and/or deletions in genome segments which are not required for multiplication of the virus.
4. Recombinantly prepared PPVs according to Claim 1 or 2, containing insertions and/or deletions in genome segments which are required for multiplication of the virus.

Images